Schimmer B P, Tsao J, Borenstein R, Endrenyi L
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
J Biol Chem. 1987 Nov 15;262(32):15521-6.
The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.
比较了Y1肾上腺皮质肿瘤细胞的福斯高林抗性突变体的腺苷酸环化酶特性与亲本Y1细胞中该酶的特性，以定位突变位点。在亲本Y1细胞中，福斯高林刺激腺苷酸环化酶活性，其动力学表明在两个位点存在相互作用；在突变细胞中，福斯高林抗性的特征是两个位点的酶活性均降低。福斯高林增强了亲本和突变体克隆中该酶对氟化钠和鸟苷-5'-基亚氨基二磷酸（Gpp(NH)p）的反应，并且突变酶对Mg2+和Mn2+的需求与亲本酶相同。与福斯高林抗性突变体相关的腺苷酸环化酶对促肾上腺皮质激素（ACTH）不敏感，并且对Gpp(NH)p的反应比亲本酶弱。在亲本Y1细胞和福斯高林抗性突变体中，霍乱毒素催化[32P]ADP-核糖从[32P]NAD+转移到与Gs的α亚基相关的三种膜蛋白中；然而，掺入突变体膜中的标记ADP-核糖量减少了多达70%。亲本和突变体膜被百日咳毒素标记的程度相同。突变型腺苷酸环化酶对ACTH和Gpp(NH)p不敏感以及突变体膜对霍乱毒素催化的ADP-核糖基化的选择性抗性表明，与Gs相关的特定缺陷参与了Y1细胞中对福斯高林抗性的突变。
